Brown Fat Cell Compositions and Methods

ABSTRACT

Methods of developing and using cell lines, such as stem cell lines, for therapeutic or cosmetic use. In one embodiment the cell lines are used to treat a wide range of degenerative and metabolic disorders including, but not limited to, obesity, diabetes, hypertension, and cardiac deficiency. Also described are methods of using such cell lines to screen for compounds that play a role in regulating a variety of processes.

RELATED APPLICATIONS

This application claims priority to and benefit of U.S. Provisionalapplication 61/502,508 filed on Jun. 29, 2011, U.S. Provisionalapplication 61/632,122 filed on Jan. 18, 2012, and U.S. Provisionalapplication 61/632,516 filed on Jan. 25, 2012 the entirety of each ofthese applications is incorporated by reference herein.

FIELD OF THE INVENTION

This invention relates to the field of cell culture and morespecifically to the culture and use of stem cells.

BACKGROUND

Brown fat is one of two types of adipose tissue found in the human body.During embryogenesis, brown fat is derived from the differentiation ofthe mesoderm. Brown fat is involved in development and homeostasis byproviding metabolic tissue capable of providing heat. Brown fat helpsregulate metabolism and nonshivering thermogenesis. In addition, brownfat plays a larger role than white adipose tissue in regulatingmetabolism. Brown fat makes up 5 percent of the body mass of a humanneonate, and less than 1 percent of the body mass of an adult.

Metabolic activity of brown fat decreases with increased body massindex. Similarly, metabolic activity of brown fat decreases withincreased body fat percent.

Stem cells have been identified and isolated in various tissues, andwhite adipose tissue stem cells have been isolated, expanded and shownto have functional therapeutic characteristics. To date, no stem cellpopulation has been identified in brown adipose tissue.

SUMMARY OF THE INVENTION

Described are methods of developing cell lines, such as stem cell lines,for therapeutic or cosmetic use. For example, the cell lines can be usedto treat a wide range of degenerative and metabolic disorders including,but not limited to, obesity, diabetes, hypertension, and cardiacdeficiency. Also described are methods of using such cell lines toscreen for compounds that play a role in regulating a variety ofprocesses, such as, but not limited to, methylation, homeostasis, andgenes involved in the regulation of metabolism, thermogenesis,activation, and/or maintenance of brown and white fat levels in thebody.

In one aspect, the invention features a method of generating a stemcell. In one embodiment the steps include obtaining brown fat tissue;isolating a stem cell from the brown fat tissue; and culturing the stemcell in a medium not comprising an animal product, thereby generating astem cell. In another embodiment the brown fat tissue is from a sampleobtained from a subject. In another embodiment the stem cell is positivefor one or more of the following cell surface markers: CD63, CD90, HLAABC, CD105, CD73, CD166, CD 9, CD44. In another embodiment the stem cellis negative for one or more of the following cell surface markers: CD34,CD19, CD86, HLA DR, Lin, CD106, CD80, CD 117. In still yet anotherembodiment the stem cell is autogeneic. In another embodiment the stemcell is allogeneic. In another embodiment the medium includes LowGlucose DMEM without phenol red, 0.5-20% Human Platelet Lysate, 1×NEAA,1× Glutamax, 1× Gentamycin, and 1000 units of Heparin.

In another aspect, the invention features a method of generating acellular extract of stem cells, progenitor cells or differentiated cellssuch as activated brown fat adipocytes (e.g., brown fat adipocytesexpressing UCP-1 and/or PRDM16) and administrating this extract into fatdeposits within the subject, thereby treating obesity.

Another aspect of the invention is a method of treating obesity in asubject. In one embodiment the method includes the steps of removingbrown fat from a subject; and administering the brown fat to a fatdeposit within the subject, thereby treating obesity in the subject. Inone embodiment the fat deposit is a white fat deposit. In anotherembodiment the fat deposit is a brown fat deposit.

In another aspect, the invention features a method of identifying acompound involved in metabolism regulation, brown fat activation and/ormaintenance, comprising contacting an autologous stem cell, progenitorcell or differentiated cell produced by a method described herein with atest compound; determining the level of metabolic activity,thermogenesis, and/or activation of certain genes such as PRDM-16 myf-5UCP-1, CYC1, NDUFA11, NDUFA13, CMT1A, ELOVL3, DIO2, LHX8, COX8A, CYFIP2,CIDEA, Cox8b, Glut4, of the stem cell, progenitor cell, ordifferentiated cell in the presence of the test compound; and comparingthe level of metabolic activity, thermogenesis and/or gene activity ofthe stem cell, progenitor cell or differentiated cell in the presence ofthe test compound to a level of metabolic activity, thermogenesis and/orgene activity of the stem cell, progenitor cell or differentiated cellin the absence of the test compound, wherein a level of metabolicactivity, thermogenesis and/or gene activity in the presence of the testcompound that is different from a level of metabolic activity,thermogenesis and/or gene activity in the absence of the test compoundidentifies the test compound as a compound involved in metabolismregulation or brown fat activation, thermogenesis and/or maintenance.

In yet another aspect the invention relates to a method of treating ametabolic disorder in a subject. In one embodiment the method includesthe steps of removing a stem cell from a donor; differentiating the stemcell into a brown adipocyte in a medium not comprising an animalproduct; and administering the brown adipocyte to a fat deposit withinthe subject, thereby treating the metabolic disorder in the subject. Inanother embodiment the metabolic disorder is selected from the groupconsisting of obesity, central obesity, diabetes, hypertension, cardiacdeficiency, ischemic cardiac disease, high blood pressure, triglyceridedyslipidemia, HDL dyslipidemia, cholesterol dyslipidemia, elevatedfasting plasma glucose, leptin dysregulation, and adipon dysregulation.In still another embodiment the brown adipocyte is administered bysubcutaneous injection. In still yet another embodiment the brownadipocyte is administered by systemic injection. In another embodimentthe medium contains a differentiation agent selected from the groupconsisting of insulin, dexamethasone, isobutylmethylxanthine, andindomethacin.

Still yet another aspect of the invention is a method of identifying acompound involved in metabolism regulation. In one embodiment the methodincludes the steps of isolating a stem cell from the brown fat tissue;culturing the stem cell in a medium not comprising an animal product;contacting the stem cell with a test compound; determining the level ofmetabolic activity of the stem cell in the presence of the testcompound; and comparing the level of metabolic activity of the stem cellin the presence of the test compound to a level of metabolic activity ofthe stem cell in the absence of the test compound, wherein a level ofmetabolic activity in the presence of the test compound that isdifferent from a level of metabolic activity in the absence of the testcompound identifies the test compound as a compound involved inmetabolism regulation. In one embodiment the determining step includesmeasuring adipogenesis. In another embodiment the method furthercomprises detecting the levels of one or more of PRDM16, BMP7, UCP1,UCP2, or MYF5. In still yet another embodiment the test compound isselected from the group consisting of protein, antibody, peptide,mutein, polynucleotide, nucleic acid aptamer, and small molecule.

An aspect of the invention relates to differentiating a stem cell into abrown fat cell. In one embodiment the method includes contacting a stemcell with a medium not comprising an animal product, thereby generatinga brown fat stem cell. In another embodiment the medium includes anagent selected from the group consisting of: insulin, dexamethasone,isobutylmethylxanthine, indomethacin, T3, rosiglitazone, FNDC5 andcombinations thereof. In still yet another embodiment the mediumincludes insulin, dexamethasone, isobutylmethylxanthine, andindomethacin. In yet another embodiment the brown fat stem cellexpresses PRDM-16, PGC-1, or a combination thereof. In still yet anotherembodiment the brown fat tissue is from a sample obtained from asubject.

BRIEF DESCRIPTION OF DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee. The present teachings described herein will bemore fully understood from the following description of variousillustrative embodiments, when read together with the accompanyingdrawings. It should be understood that the drawings described below arefor illustration purposes only and are not intended to limit the scopeof the present teachings in any way.

FIG. 1 is a schematic diagram depicting exemplary methods of injectingbrown fat cells or brown fat precursor cells into a subject.

FIG. 2 is a micrograph of a cell culture ready for passaging.

FIG. 3 are images showing gene expression by RT-PCR in white adiposedepots and brown adipose depots.

FIGS. 4 A and B are fluorescent micrographs of cells stained with stemcell markers.

FIGS. 5A-F is a series of graphs showing detection by flow cytometry ofvarious cell type markers.

FIG. 6 is a micrograph showing presence of fat droplets in cell culture.

FIGS. 7A and B are micrographs of cells stained with oil red 0. FIG. 7Ashows large fat droplets (200 μm) and FIG. 7B shows small fat droplets(50 μm).

FIGS. 8A and B are micrographs of cells stained with markers ofdifferentiation. FIG. 8A shows staining with alizarin red, a marker ofosteogenesis. FIG. 8B shows staining with osteocalcin antibody andalcian blue, to show chondrogenesis.

FIG. 9 is a scanning electron micrograph of exosomes isolated from brownfat cells.

FIG. 10 shows a timecourse of glucose levels and body weight in micetreated with brown fat progenitor cells.

FIG. 11 shows a timecourse of triglyceride levels and cholesterol levelsin mice treated with brown fat progenitor cells.

FIG. 12 shows a timecourse of leptin levels and adipon levels in micetreated with brown fat progenitor cells.

DETAILED DESCRIPTION

The invention is based, at least in part, on the discovery that cells,such as autologous or nonautologous brown fat cells or stem cells, canbe isolated and used to modify metabolism and/or thermogenesis in asubject, and/or to treat a wide range of disorders in a subject, such asmetabolic disorders. (FIG. 1)

As described herein, brown fat cells and brown fat stem cells can beused therapeutically to treat a number of metabolic disorders. In someembodiments, the metabolic disorder is selected from central obesity(waist circumference of 40 inches or greater in males or 36 inches orgreater in females); triglyceride dyslipidemia (1.7 mmol/L (150 mg/dl)or greater); HDL dyslipidemia (less than 40 mg/dl in males, less than 50mg/dl in females); blood pressure (130/85 mmHg or greater); and elevatedfasting plasma glucose (6.1 mmol/L (110 mg/dl) or greater).

The cells isolated can be from, for example, an autologous source or anallogeneic source. However, other sources also can be used.

Cellular products can be made from these cultured stem cells derivedfrom mediastinal fat depots, such as cellular extracts. These cellularextracts then can be used therapeutically.

Cells or stem cells described in this application isolated from brownfat depots also can be used to screen compounds (including naturallyoccurring compounds) that increase the levels of brown fat, metabolism,and or gene expression profiles that favor higher metabolic activity.Similarly, cells or stem cells described herein can be used to identifycompounds (including naturally occurring compounds) that decrease thelevels of brown fat, metabolism, and or gene expression profiles thatdisfavor higher metabolic activity. In some embodiments, the screeningis small molecule screening.

As used herein, “treatment” means any manner in which one or more of thesymptoms of a disease or disorder are ameliorated or otherwisebeneficially altered. As used herein, amelioration of the symptoms of aparticular disorder refers to any lessening, whether permanent ortemporary, lasting or transient, of the symptoms, which can beattributed to or associated with treatment by the compositions andmethods of the present invention.

The terms “effective amount” and “effective to treat,” as used herein,refer to an amount or a concentration of one or more of the compositionsdescribed herein utilized for a period of time (including acute orchronic administration and periodic or continuous administration) thatis effective within the context of its administration for causing anintended effect or physiological outcome.

As used herein, the term “subject” means an animal, human or non-human,to whom treatment according to the methods of the present disclosure isprovided. Veterinary and non-veterinary applications are contemplated.The term includes, but is not limited to, mammals. Typical subjectsinclude humans, farm animals, and domestic pets such as cats and dogs.In some embodiments, the subject is a mammal. In some embodiments, thesubject is a human subject, e.g., an obese human subject. In someembodiments, the subject is a non-human mammal, e.g., an experimentalanimal, a companion animal, or an animal that is raised for food.

As used herein, an “isolated” or “purified” cell is a cell substantiallyfree of contaminating components from a cell culture or tissue sourcefrom which the cell is derived. “Substantially free” means that apreparation of a selected cell has less than about 50%, (e.g., less thanabout 40%, 30%, 20%, or 10%) of non-selected components. Such anon-selected component is also referred to herein as “contaminatingcomponent.” When the isolated cells are recombinantly produced, they canbe substantially free of culture medium, i.e., culture medium representsless than about 20%, (e.g., less than about 10% or 5%) of the volume ofthe cell preparation.

As used herein, “stem cells” are cells capable of both self-renewal anddifferentiation into many different cell lineages (is pluripotent).“Progenitor cells” refers to a subset of stem cells with phenotypessimilar to that of a stem cell. A progenitor cell is capable ofself-renewal and is typically multipotent. As used herein,“differentiated cells” refers to a subset of cells with phenotypes of amature cell type specific to a particular tissue or organ system.

Methods of Obtaining Cells

In general, cells useful in the methods described herein can be obtainedfrom a subject, such as by isolating the cells from a subject, orclinical grade embryonic stem cells can be obtained from a commercialsource. In certain embodiments, the cells are part of a tissue sample,such as a brown fat sample, from a subject. The tissue sample can beadministered directly into a subject or the cells can be isolated fromthe tissue and processed as described herein. In other embodiments, thecells are stem cells, such as from a bone marrow biopsy, and can beprocessed as described herein.

Certain cells that can be used in the methods described herein areunipotent, multipotent, or pluripotent cells and can be of mesodermalorigin. In some embodiments, the cells are brown adipose progenitorcells, mature brown fat cells or stem cells. Brown adipose progenitorcells, mature brown fat cells or stem cells can be identified bydetermining the presence or absence of one or more cell surfaceexpression markers. Exemplary cell surface markers that can be used toidentify a brown adipose progenitor cell, mature brown fat cell or stemcell include, but are not limited to, MYF5, UCP-1, PRDM-16, SSEA-4,Sca-1, CD45, Mac-1, CD29 (integrin (31), CD105 (Endoglin), CD166(ALCAM), desmin, vimentin, and c-kit. In yet other methods, the cellsare brown adipose cells.

In any of the methods described herein, the cells can be autologous,syngeneic, allogeneic, or xenogeneic.

Cells suitable for use in the methods described herein can be found in avariety of tissues and organs including, but not limited to, forexample, skeletal muscle, cardiac muscle, smooth muscle, prostate,dermis, the cardiovascular system, mammary gland, liver, neonatal skin,calvaria, bone marrow, the intestine, adipose tissue (e.g., whiteadipose tissue, brown adipose tissue), peripheral blood, mobilizedperipheral blood, and umbilical cord. Cells can be isolated from suchtissues and organs in a number of known ways, including, e.g., biopsy,apheresis, or liposuction.

In some embodiments, cells are obtained from adipose tissue, e.g., whiteor brown adipose deposits. For example, brown adipose tissue can beharvested using known means (e.g., by biopsy) from specific anatomicalregions of a subject, such as cervical-supraclavicular regions, superiormediastinal regions, and regions superficial or lateral tosternocleidomastoid muscles. Brown adipose tissue can be identified in anumber of ways, such as by imaging (e.g., by PET scan). The brownadipose tissue can be subjected to minimal processing (such as bywashing with buffer, e.g., DPBS, and mechanically separated intoportions) and administered into a subject as described herein.

In other methods, cells, such as brown fat cells, mononuclear cells,progenitor cells, or stem cells, can be isolated from contaminatingcomponents of the tissue sample. For example, the tissue sample can betreated with an enzyme such as collagenase (e.g., Type I, II, or III),dispase, hyaluronidase, or elastase and cells can be isolated byfiltration or centrifugation. Alternatively, the tissue sample can betreated chemically, such as with EDTA, and cells can be isolated using,e.g., mechanical disruption (e.g., vortexing). The isolated cells can bewashed with a suitable buffer, such as DPBS.

In some methods, cells are isolated from a bone marrow sample of asubject. For example, a bone marrow sample can be obtained using needleaspiration or other known technique. In certain instances, cells can beisolated from a bone marrow sample using a Ficoll-Hypaq densitygradient.

In yet other methods, cells are isolated from skin of a subject. Forexample, a punch biopsy can be used to obtain a skin sample. In oneexemplary method, a punch biopsy is used to obtain a 0.5 cm piece ofskin, which is washed three times with DPBS, and the dermis is removedfrom the biopsy. The skin is then cut into small (about 3 mm) sectionsand plated onto the wells of a 6-well tissue culture plate with asterile cover slip over the tissue and the cells cultured.

Methods of Culturing Cells

In certain methods, cells obtained as described herein are maintained ina suitable culture medium, e.g., a culture medium not comprising ananimal-based product (such as bovine serum or calf serum) in order toobtain a larger population of cells. The culture methods can includeallowing the cells to undergo sufficient rounds of doubling, e.g., toproduce either a clonal cell strain or a heterogeneous cell strain ofdesired size, e.g., a sufficient number to provide a therapeutic effectto a subject, or a sufficient number to establish a stable cell line.For example, cells can be cultured at 37° C. for a period of 3-6 weeksafter an initial biopsy.

The culture media used in the methods described herein do not includeanimal-based products. An exemplary culture medium suitable for themethods described herein can include human platelet lysate and ananticoagulant, such as heparin or acid-citrate-dextrose. Anotherexemplary culture medium includes human pooled AB type serum and ananticoagulant. Other examples of culture medium can include ModifiedEagle Medium (MEM) (such as Dulbecco's MEM or alpha MEM) supplementedwith glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid,L-proline, L-serine, L-glutamine, and an antibiotic. One exemplaryculture medium includes: DMEM (low glucose, without phenol red); 0.5-20%human platelet lysate; 1× non-essential amino acids (NEAA); 1× Glutamax;1× gentamycin, and 1000 units of heparin.

In some embodiments, the cells can be maintained in the culture mediumunder standard conditions, such as described in, e.g., Freshney (1994)Culture of Animal Cells, a Manual of Basic Technique, third edition,Wiley-Liss, New York.

In certain embodiments, the cells are cultured in human platelet lysateor human pooled AB type serum derived from subjects who have beenexposed to cold temperatures, e.g., a temperature suitable to generate asympathetic nervous system response in the subject.

In other embodiments, the cells can be maintained in a hypoxicenvironment (e.g., 0 to about 5% O₂ at 37° C.).

In certain embodiments, cultured cells can express MYF-5, BMP7, and/orPRDM16, SSEA4. In other embodiments, cells cultured from muscle tissuecan express SCAT and not express c-Kit or CD45.

Methods of Differentiating Cells

In certain embodiments, cells obtained as described herein aremaintained in a suitable differentiation medium, e.g., a medium notcomprising an animal-based product. For example, stem cells can bemaintained in a differentiation medium for a time sufficient to resultin the differentiation of the stem cells into brown adipocyte progenitorcells or brown adipocytes. In particular embodiments, cells aremaintained in a differentiation medium for a time sufficient to resultin the differentiation of the stem cells into MYF5 expressing cells. Incertain instances, cells are maintained in the differentiation mediumfor, e.g., 1-5 days, 5-10 days, 10-14 days, 14-21 days, 21-28 days, orlonger.

In some embodiments, the differentiation medium includes one or morechemical or hormone differentiation inducers and/or thiazolidinediones(as described in, e.g., Klien et al., J. Biol. Chem. 274:34795-34802(1999); Hauner et al., J. Clin. Invest. 84:1663-1670 (1989)). In someinstances, the differentiation medium includes insulin, dexamethasone,isobutylmethylxanthine, and indomethacin and rosiglitazone. Oneexemplary differentiation medium includes DMEM (low glucose withoutphenol red); 0.5%-20% Human Platelet Lysate; 1×NEAA; 1× Glutamax; 1×gentamycin; 1000 units of heparin; 10 ug/mL of insulin; 1 uMdexamethasone; 200 uM indomethacin; and 0.5 mM isobutylmethylxanthine.In another embodiment, the differentiation medium includes BMP7. Inanother embodiment, the differentiation medium includes DMEM (lowglucose without phenol red); 0.5%-20% Human Platelet Lysate; 1×NEAA; 1×Glutamax; 1× gentamycin; 1000 units of heparin; 0.5 mMisobutylmethylxanthine, 125 nM indomethacin, 5 uM dexamethosaone, 850 nMinsulin, 1 nM T3, and 1 uM rosiglitazone. In another embodiment, thedifferentiation medium includes DMEM (low glucose without phenol red);0.5%-20% Human Platelet Lysate; 1×NEAA; 1× Glutamax; 1× gentamycin; 1000units of heparin; 0.5 mM isobutylmethylxanthine, 125 nM indomethacin, 5uM dexamethosaone, 850 nM insulin, 1 nM T3, and 1 uM rosiglitazone and20 nM FNDC5.

In some instances, the cells are maintained in differentiation mediumuntil one or more markers of brown adipocyte differentiation aredetected. Non-limiting examples of markers of differentiation includethe expression of cell death-inducing DFF45-like effector A (CIDEA),Type II deiodinaie, PPAR gamma coactivator (PGC)-1 alpha, PGC-1 beta,uncoupling protein (e.g., UCP1), PRDM16, or CIG30, PRDM-16, CYC1,NDUFA11, NDUFA13, CMT1A, ELOVL3, DIO2, LHX8, COX8A, CYFIP2, Cox8b,Glut4.

In some embodiments, the methods include exposing the cells to coldshock by culturing or differentiating the cells during daily cycles of4° C.-25 C for one hour followed by culture at standard conditions (37°C., 5% CO₂ or hypoxic conditions).

In some embodiments, the methods include evaluating the level ofadipogenesis following maintenance in a differentiation medium.Adipogenesis can be evaluated by measuring, e.g., lipid accumulation(e.g., using oil red-o (ORO) staining), cell morphology (e.g., usingvisual, e.g., microscopic, inspection of the cells), or cellthermodynamics (e.g., cytochrome oxidase activity, Na+-K+-ATPase enzymeunits, or other enzymes involved in brown adipocyte thermogenesis). Inaddition, in some embodiments, functional brown fat adipogenesisfollowing differentiation can be determined by fatty acid uptake assaysor oxygen consumption rate.

Further Processing Methods

In some embodiments, cells obtained as described herein can be subjectedto additional processing before administration into a subject. Incertain embodiments, a cell obtained as described herein can berecombinantly modified to express one or more genes, e.g., a geneinvolved in brown adipogenesis. For example, a cell can be transfectedwith one or more nucleic acids encoding DFF45-like effector A (CIDEA),Type II deiodinaie, PPAR gamma coactivator (PGC)-1 alpha, PGC-1 beta,uncoupling protein (e.g., UCP1), PRDM16, or CIG30, PRDM-16, CYC1,NDUFA11, NDUFA13, CMT1A, ELOVL3, DIO2, LHX8, COX8A, CYFIP2, Cox8b, Glut4or combinations of the genes, prior to administration into a subject.

In other embodiments, the cells can be pluripotent stem cellsartificially derived (e.g., differentiated or partially differentiated)from a non-pluripotent cell. For example, skin cells, such as dermalfibroblasts, can be reprogrammed to a pluripotent state by transfectionwith OCT4, Nanog, or SSEA4. Such cells are known in the art as inducedpluripotent stem cells. In other embodiments the cells can be furthermanipulated after becoming pluripotent by transfection with one or morenucleic acids encoding DFF45-like effector A (CIDEA), Type IIdeiodinaie, PPAR gamma coactivator (PGC)-1 alpha, PGC-1 beta, uncouplingprotein (e.g., UCP1), PRDM16, or CIG30, PRDM-16, CYC1, NDUFA11, NDUFA13,CMT1A, ELOVL3, DIO2, LHX8, COX8A, CYFIP2, Cox8b, Glut4 or combinationsof the genes, (ie. PPARG2, CEBPB, PRDM16)

In further embodiments, a cell can be mitotically inactivated prior toadministration into a subject. Without wishing to be bound by theory, itis believed that upon administration to a subject, cells obtained and/ortreated as described herein express certain factors that may have aparacrine effect on surrounding cells and tissues. Accordingly, mitoticinactivation prevents further cell division while allowing theinactivated cells to maintain the paracrine effect. In certainembodiments, cells can be treated with chemical or gamma irradiationsufficient to mitotically inactivate the cells.

In some embodiments, a cell extract or lysate can be prepared from acell described herein and administered to a subject. For example, cellscan be obtained and cultured, and about 10⁶ to about 10¹⁰ cells can becollected and used to prepare a cell-free extract using known methods.In one exemplary method, an extract is prepared by subjecting thecollected cells to freeze-thaw cycles (e.g., 1, 2, 3, 4, 5, or morefreeze-thaw cycles) using an ethanol/dry ice bath. The extract is thencentrifuged (e.g., at about 14,000 rpm) to remove insoluble material.The extract can then be administered to a subject.

Expression Methods

In certain embodiments, a cell described herein can be recombinantlymodified to express one or more genes. Such nucleic acids can beincorporated into an expression vector. Expression vectors comprising anucleic acid sequence described herein can be administered in anyeffective carrier, e.g., any formulation or composition capable ofeffectively delivering the component gene to cells in vivo. Approachesinclude insertion of the gene in viral vectors, including recombinantretroviruses, adenovirus, adeno-associated virus, lentivirus, poxvirus,alphavirus, and herpes simplex virus-1, or recombinant bacterial oreukaryotic plasmids. Viral vectors transfect cells directly; plasmid DNAcan be delivered naked or with the help of, for example, cationicliposomes (lipofectamine) or derivatized (e.g., antibody conjugated),polylysine conjugates, gramicidin S, artificial viral envelopes or othersuch intracellular carriers, as well as direct injection of the geneconstruct or CaPO₄ precipitation carried out in vivo.

In some embodiments, the expression vector is a viral vector containingone or more nucleic acid sequences (e.g., cDNA). Retrovirus vectors canbe used as a recombinant gene delivery system for the transfer ofexogenous genes in vivo, particularly into humans as is known to oneskilled in the art. (Protocols for producing recombinant retrovirusesand for infecting cells in vitro or in vivo with such viruses can befound in Ausubel, et al., eds., Current Protocols in Molecular Biology,Greene Publishing Associates (1989), Sections 9.10-9.14, and otherstandard laboratory manuals. Another viral gene delivery system usefulin the present methods utilizes adenovirus-derived. Yet another viralvector system useful for delivery of nucleic acids is theadeno-associated virus (AAV) as is known to one skilled in the art.

In addition to viral transfer methods, such as those illustrated above,non-viral methods can also be employed to express a nucleic acid into acell described herein. Typically non-viral methods of gene transfer relyon the normal mechanisms used by mammalian cells for the uptake andintracellular transport of macromolecules. In some embodiments,non-viral gene delivery systems can rely on endocytic pathways for theuptake of the subject gene by the targeted cell. Exemplary gene deliverysystems of this type include liposomal derived systems, poly-cationicconjugates such as polyamine and polylysine, and artificial viralenvelopes. Other embodiments include plasmid injection systems as knownto one skilled in the art. Other non-viral vectors include ascaffold/matrix attached region (S/MAR)-based vector. In particularembodiments, a cell described herein is transfected with an S/MAR-PRDM16construct, an S/MAR-BMP-7/PRDM16 construct, or an S/MAR-BMP-7 construct.In another embodiment the cell is transfected with an S/MAR constructcontaining one or more nucleic acids encoding DFF45-like effector A(CIDEA), Type II deiodinaie, PPAR gamma coactivator (PGC)-1 alpha, PGC-1beta, uncoupling protein (e.g., UCP1), PRDM16, or CIG30, PRDM-16, CYC1,NDUFA11, NDUFA13, CMT1A, ELOVL3, DIO2, LHX8, COX8A, CYFIP2, Cox8b, Glut4or combinations of the genes or a combination of them (i.e. PPARG2,CEBPB, PRDM16).

In some embodiments, a nucleic acid can be expressed using naked DNAconstructs and/or DNA vector based constructs as is known to one skilledin the art. In some embodiments, DNA vectors can be introduced intotarget cells via conventional transformation or transfection techniques.As used herein, the terms “transformation” and “transfection” areintended to refer to a variety of art-recognized techniques forintroducing foreign nucleic acid (e.g., DNA) into a target cell,including calcium phosphate or calcium chloride co-precipitation,DEAE-dextran-mediated transfection, lipofection, electroporation, genegun, sonoporation, or magnetofection.

All the molecular biological techniques required to generate anexpression construct described herein are standard techniques that willbe appreciated by one of skill in the art.

Methods of Administration

Methods described herein can include implanting tissue or cells, e.g.,cells obtained or isolated as described herein, into a subject to betreated. The cells can be differentiated brown adipocytes (e.g.,isolated brown adipocytes or differentiated adipocytes produced asdescribed herein), or can be stem cells or undifferentiated cells, whichcells, or their progeny (i.e., daughter cells), will differentiate intobrown adipocytes after implantation. These methods are useful, e.g., formodifying metabolism in a subject as described herein.

Prior to administration into a subject, the cells can be washed (e.g.,in isotonic PBS) to remove any contaminants, including contaminatingcomponents of tissue sample, culture media or differentiation media,before implantation. The number of required cells is variable anddepends on a variety of factors, including but not limited to, the celltype used, the site of implantation of the cells (for example, thenumber of cells that can be used can be limited by the anatomical siteof implantation), and the age, surface area, and clinical condition ofthe subject. In some embodiments, at least about 10⁵, 10⁶, 10⁷, 10⁸,10⁹, or about 10¹⁰ cells are implanted into the subject.

Methods for implanting cells within a subject are known in the art,e.g., using a delivery system configured to allow the introduction ofcells into a subject. In general, the delivery system can include areservoir containing cells and a needle in fluid communication with thereservoir. Such delivery systems are also within the scope of theinvention. Generally, such delivery systems are maintained in a sterilemanner. Various routes of administration and various sites (e.g., renalsub capsular, subcutaneous, central nervous system (includingintrathecal), intravascular, intrahepatic, intrasplanchnic,intraperitoneal (including intraomental), intramuscularly implantation)can be used.

The cells can be in a pharmaceutically acceptable carrier, with orwithout a scaffold, matrix, or other implantable device to which thecells can attach (examples include carriers made of, e.g., collagen,fibronectin, elastin, cellulose acetate, cellulose nitrate,polysaccharide, fibrin, gelatin, and combinations thereof). Initially1,000,000 cells where seeded onto porous extracellular scaffolds andcultured for 5 days. Differentiation into brown adipose was initiated byadding DMEM (low glucose without phenol red) containing

10% Human Platelet Lysate; 1X NEAA; 1X Glutamax; 1X gentamycin; 1000units of heparin; 0.5 mM isobutylmethylxanthine, 125 nM indomethacin, 5uM dexamethosaone, 850 nM insulin, 1 nM T3, 1 uM rosiglitazone.and the cells were grown in this medium for 2 days followed by furtherdifferentiation for an additional 18 days in media composed of:

DMEM (low glucose without phenol red); 10% Human Platelet Lysate; 1XNEAA; 1X Glutamax; 1X gentamycin; 1000 units of heparin; 0.5 mMisobutylmethylxanthine, 125 nM indomethacin, 5 uM dexamethosaone, 850 nMinsulin, 1 nM T3, 1 uM rosiglitazone 20 nM FNDC5.

Cells implanted using scaffolding were shown under scanning electronmicroscopy to attach to the scaffold. Further measurements of fatty aciduptake by the cells showed that the cells were metabolically active.

In particular instances, the cells are implanted into white fat or brownfat in the subject, and/or into or near an anatomical region containingwhite fat or brown fat.

Where non-immunologically compatible cells are used (e.g., wherenon-autologous cells are administered to a subject), animmunosuppressive compound, e.g., a drug or antibody, can beadministered to the subject at a dosage sufficient to achieve inhibitionof rejection of the cells. Dosage ranges for immunosuppressive drugs areknown in the art. Dosage values may vary according to factors such asthe disease state, age, sex, and weight of the individual.

Subjects

The methods and compositions described herein are useful for thetreatment of metabolic disorders. Generally, the methods includeadministering an effective amount of cells described herein to a subjectin need thereof, including a subject that has been diagnosed to be inneed of such treatment. Subjects can include mammals

In some embodiments, the methods include identifying a subject in needof treatment (e.g., a subject having or at risk of developing ametabolic disorder), and administering to the subject an effectiveamount of tissue or cells described herein. In certain instances, thesubject is diagnosed as being an overweight or obese subject, e.g., witha body mass index (BMI) of 25-29 or 30 or above or a subject with aweight related disorder. A subject in need of treatment with the methodsdescribed herein can be selected based on the subject's body weight orbody mass index. In some embodiments, the methods include evaluating thesubject for one or more of: weight, adipose tissue stores, adiposetissue morphology, insulin levels, insulin metabolism, glucose levels,thermogenic capacity, and cold sensitivity. In some embodiments, subjectselection can include assessing the amount or activity of brown adiposetissue in the subject and recording these observations.

The evaluation can be performed before, during, and/or after theadministration of the cells described herein. For example, theevaluation can be performed at least 1 day, 2 days, 4, 7, 14, 21, 30 ormore days before and/or after the administration of cells describedherein.

Metabolism and Metabolic Disorders

Metabolism is a cascade of chemical reactions that regulate themechanisms by which living organisms regulate, maintain and respond tointrinsic and extrinsic factors that affect their ability to maintain,grow, and reproduce. An imbalance of this metabolic activity can lead todownstream cellular events that can give rise to a number ofdegenerative disorders. Such disorders can bring about not only symptomsof a wide range of diseases, but can interfere with proper methylationof the imprinting pattern of male and female germ cells, thusestablishing a genetic predisposition in the new generation todegenerative disorders.

In certain embodiments, the tissue or cells described herein areadministered to a subject to modify, e.g., increase, metabolic activityin the subject. In certain embodiments, the subject has a disorder suchas, but not limited to, obesity, osteoarthritis, hypertension, diabetes,an auto-immune disorder, stroke, kidney failure, neoplasia, or a cardiacdeficiency such as ischemic heart disease. In particular instances, theadministration of cells described herein treat the disorder.

In other embodiments, subjects can be screened for levels ofadipogenesis markers, such as DFF45-like effector A (CIDEA), Type IIdeiodinaie, PPAR gamma coactivator (PGC)-1 alpha, PGC-1 beta, uncouplingprotein (e.g., UCP1), PRDM16, or CIG30, PRDM-16, CYC1, NDUFA11, NDUFA13,CMT1A, ELOVL3, DIO2, LHX8, COX8A, CYFIP2, Cox8b, Glut4, and/or BMP7, andthe information used as a diagnostic tool for obesity, hypertension,diabetes or ischemic cardiac disease. For example, an adipose tissuesample or a blood sample can be obtained from a subject and the level ofDFF45-like effector A (CIDEA), Type II deiodinaie, PPAR gammacoactivator (PGC)-1 alpha, PGC-1 beta, uncoupling protein (e.g., UCP1),PRDM16, or CIG30, PRDM-16, CYC1, NDUFA11, NDUFA13, CMT1A, ELOVL3, DIO2,LHX8, COX8A, CYFIP2, Cox8b, Glut4, and/or BMP7 can be measured in thesample. A level of DFF45-like effector A (CIDEA), Type II deiodinaie,PPAR gamma coactivator (PGC)-1 alpha, PGC-1 beta, uncoupling protein(e.g., UCP1), PRDM16, or CIG30, PRDM-16, CYC1, NDUFA11, NDUFA13, CMT1A,ELOVL3, DIO2, LHX8, COX8A, CYFIP2, Cox8b, Glut4, and/or BMP7 below apredetermined level indicates the subject has, or is at risk ofdeveloping, a metabolic disorder, such as obesity, hypertension,diabetes or ischemic cardiac disease. The predetermined level can be thelevel of a marker in a corresponding sample from a subject not havingthe metabolic disorder. In yet other embodiments, the levels of brownfat and white fat in a subject can be measured, e.g., using PET scan,and a ratio of brown fat to white fat can be determined. A ratio ofbrown fat to white fat less than a predetermined level can indicate thesubject has, or is at risk of developing, a metabolic disorder, such asobesity, hypertension, diabetes or ischemic cardiac disease.

Methods of Screening for Adipogenesis Compounds

Cells isolated or cultured as described herein can be used to screen forcompounds that modify, e.g., increase or reduce, metabolic activity. Insome embodiments, a cell, e.g., a brown adipocyte progenitor cell,differentiated cell or stem cell, can be contacted with a test compoundand the level of a marker for adipogenesis, e.g., a marker describedherein (e.g., PRDM16, BMP7, UCP1 or MYF5) can be measured. A testcompound that increases the level of a marker for adipogenesis relativeto a cell not contacted with the test compound is identified as acompound that increases metabolic activity, whereas a test compound thatreduces a marker for adipogenesis is identified as a compound thatdecreases metabolic activity. In another embodiment the cell is a whiteadipocyte and it is transfected with a construct expressing a reportergene (i.e. GFP, luciferase) under the control of DFF45-like effector A(CIDEA), Type II deiodinaie, PPAR gamma coactivator (PGC)-1 alpha, PGC-1beta, uncoupling protein (e.g., UCP1), PRDM16, or CIG30, PRDM-16, CYC1,NDUFA11, NDUFA13, CMT1A, ELOVL3, DIO2, LHX8, COX8A, CYFIP2, Cox8b, Glut4or a combination of them. A test compound that increases the level of amarker for adipogenesis relative to a cell not contacted with the testcompound is identified as a compound that increases metabolic activity,whereas a test compound that reduces a marker for adipogenesis isidentified as a compound that decreases metabolic activity

In one exemplary method, a cell described herein is transfected with anexpression vector that includes a nucleic acid encoding DFF45-likeeffector A (CIDEA), Type II deiodinaie, PPAR gamma coactivator (PGC)-1alpha, PGC-1 beta, uncoupling protein (e.g., UCP1), PRDM16, or CIG30,PRDM-16, CYC1, NDUFA11, NDUFA13, CMT1A, ELOVL3, DIO2, LHX8, COX8A,CYFIP2, Cox8b, Glut4, BMP7, MYF5 or a combination of them, under thecontrol of an inducible promoter. Nonlimiting examples of such promotersinclude chemically-regulated promoters (i.e. tetracycline, steroids andmetals), or temperature specific promoters that will activate andpromote expression at a permissive temperature, such as below normalbody temperature of a subject.

In one example, a temperature specific promoter is placed in a viral ornon viral vector upstream of a nucleic acid encoding DFF45-like effectorA (CIDEA), Type II deiodinaie, PPAR gamma coactivator (PGC)-1 alpha,PGC-1 beta, uncoupling protein (e.g., UCP1), PRDM16, or CIG30, PRDM-16,CYC1, NDUFA11, NDUFA13, CMT1A, ELOVL3, DIO2, LHX8, COX8A, CYFIP2, Cox8b,Glut4 or a combination of them, MYF5 and/or BMP7. A cell isolated fromadipose tissue (white or brown), bone marrow, skin, muscle, or umbilicalcord is transfected with the vector. The cell is then cultured under thepermissive temperature (which allows gene expression from the promoter)in the presence or absence of a test compound, and the effect of thetest compound on adipogenesis is determined.

Test compounds include, e.g., proteins (including antibodies), muteins,polynucleotides, nucleic acid aptamers, hormones (i.e. Irisin) andpeptide and nonpeptide small organic molecules. Test compounds can beisolated from natural sources, prepared synthetically or recombinantly,or any combination of the same. Particular, nonlimiting examples of testcompounds include 2,4-dinitrophenol, Ephedrine, Sibutramine, FGF21, Bileacids, and Beta-3 adrenergic receptor agonists.

All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Inaddition, the materials, methods, and examples are illustrative only andnot intended to be limiting. Unless otherwise defined, all technical andscientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art to which this inventionbelongs. Although methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of the presentinvention, suitable methods and materials are described herein.

The disclosure is further illustrated by the following examples. Theexamples are provided for illustrative purposes only. They are not to beconstrued as limiting the scope or content of the disclosure in any way.

EXAMPLES Example 1 Brown Fat Isolation Processing

In one example, a patient first underwent a ¹⁸F-fluorodeoxyglucose(¹⁸F-FDG) PET-CT scan to identify brown fat deposits. Prior toundergoing the PET-CT scan, the patient was exposed to temperaturesranging from 1° C.-25° C. (to increase the uptake of ¹⁸F-FDG) for 1-2hours per day for a period of one month. Deposits identified within thecervical-supraclavicular and mediastinal area were biopsied using aneedle. Brown fat biopsies were washed three times in DPBS (−) andeither treated with 0.075%-0.2% Collagenase Type IA with vigorousshaking at 37° C. for 30-60 minutes or treated with EDTA for 30 minutesand subjected mechanical tissue disruption for 5-30 minutes. Thetissue/cells were washed three times with DPBS (−), and 1×10⁶-1×10¹⁰cells were injected into white fat deposits in the patient. Optionally,following enzymatic or mechanical tissue/cell isolation, thetissue/cells were exposed to cold temperature (0° C.-4° C.) for 1-48hours prior to injection.

Example 2 Brown Fat Culture

In this example, cells were cultured prior to injection into a patient.The cells obtained from Example 1 were plated into hyper flasks at aconcentration of 1000 cells/cm² in a culture medium comprising in thisembodiment:

DMEM Low Glucose without phenol red 0.5-20% Human Platelet Lysate 1XNEAA 1X Glutamax 1X Gentamycin 1000 units of Heparin

The cells were cultured for 3-6 weeks. The cells were then characterizedby flow-cytometry and analyzed for the expression of one or more of thefollowing markers: SCA-1, CD34, SSEA1, SSEA4, OCT4, CD31, Wnt5a,Telomerase activity, alpha-SMA, STRO-1, MYF5, PRDM16, or UCP1. The cellswere then expanded to concentrations of 1×10⁶-1×10¹⁰ for implantationinto white fat deposits of the patient.

Example 3 Differentiation of Cells into Brown Fat Cells

In this example, cells (such as stem cells or precursor brownadipocytes) obtained from a brown fat deposit of a patient weredifferentiated and subsequently injected into a patient. The cellsobtained from Example 1 were plated out at 1000 cells/cm² in hyperflasks containing a differentiation medium comprising, in thisembodiment:

DMEM Low Glucose with out phenol red 0.5-20% Human Platelet Lysate 1XNEAA 1X Glutamax 1X Gentamycin 1000 units of Heparin 10 ug/mL of insulin1 uM Dexamethasone 200 uM Indomethacin 0.5 mM Isobutylmethylxanthine

In an alternative embodiment the differentiation medium included:

DMEM (low glucose without phenol red) 0.5%-20% Human Platelet Lysate; 1XNEAA; 1X Glutamax; 1X gentamycin; 1000 units of heparin; 0.5 mMisobutylmethylxanthine, 125 nM indomethacin, 5 uM dexamethosaone, 850 nMinsulin, 1 nM T3, 1 uM rosiglitazone.and the cells were grown in this medium for 2-6 days followed by furtherdifferentiation for an additional 6-21 days in media composed of:

DMEM (low glucose without phenol red); 0.5%-20% Human Platelet Lysate;1X NEAA; 1X Glutamax; 1X gentamycin; 1000 units of heparin; 0.5 mMisobutylmethylxanthine, 125 nM indomethacin, 5 uM dexamethosaone, 850 nMinsulin, 1 nM T3, 1 uM rosiglitazone 20 nM FNDC5.

After being maintained in the differentiation medium for 10-30 days,cells were characterized by expression of DFF45-like effector A (CIDEA),Type II deiodinaie, PPAR gamma coactivator (PGC)-1 alpha, PGC-1 beta,uncoupling protein (e.g., UCP1), PRDM16, or CIG30, PRDM-16, CYC1,NDUFA11, NDUFA13, CMT1A, ELOVL3, DIO2, LHX8, COX8A, CYFIP2, Cox8b,Glut4. 1×10⁶-1×10¹⁰ of the cells are then prepared for injection intothe patient.

Example 4 Culturing and Differentiating Embryonic Stem Cells

In this example clinical grade embryonic stem cells were first grown andexpanded. The cells were then differentiated into brown fatpre-adipocytes or fully differentiated brown fat adipocytes by exposingthe cells, in one embodiment, to the following media:

DMEM Low Glucose with out phenol red 0.5-20% Human Platelet Lysate 1XNEAA 1X Glutamax 1X Gentamycin 1000 units of Heparin 10 ug/mL of insulin1 uM Dexamethasone 200 uM Indomethacin 0.5 mM Isobutylmethylxanthine

In another embodiment, the cells were grown for 2-6 days in a mediumcomprising:

DMEM (low glucose without phenol red); 0.5%-20% Human Platelet Lysate;1X NEAA; 1X Glutamax; 1X gentamycin; 1000 units of heparin; 0.5 mMisobutylmethylxanthine, 125 nM indomethacin, 5 uM dexamethosaone, 850 nMinsulin, 1 nM T3, 1 uM rosiglitazone.

This is followed by further differentiation for an additional 6-21 daysin media composed of:

DMEM (low glucose without phenol red); 0.5%-20% Human Platelet Lysate;1X NEAA; 1X Glutamax; 1X gentamycin; 1000 units of heparin; 0.5 mMisobutylmethylxanthine, 125 nM indomethacin, 5 uM dexamethosaone, 850 nMinsulin, 1 nM T3, 1 uM rosiglitazone, 20 nM FNDC5.

Optionally, the cells were transfected, prior to differentiation orafter differentiation, with a non-viral vector (scaffold/matrix attachedregion (S/MAR)) with a PRDM-16 gene by itself or in tandem with BMP-7 orMYF-5 or DFF45-like effector A (CIDEA), Type II deiodinaie, PPAR gammacoactivator (PGC)-1 alpha, PGC-1 beta, uncoupling protein (e.g., UCP1),PRDM16, or CIG30, PRDM-16, CYC1, NDUFA11, NDUFA13, CMT1A, ELOVL3, DIO2,LHX8, COX8A, CYFIP2, Cox8b, Glut4 or a combination of them driven by astrong promoter such as CAGGS or PGK. The cells were then inactivatedwith a chemical based method such as mitomycin C or gamma irradiationand implanted into a patient at concentrations of 1×10⁶-1×10¹⁰ cells.

Example 5 Brown Fat Human Depot Biopsy

In this example a patient underwent ¹⁸F-PET-CT scan to identify tissuethat has metabolic activity. An area with high metabolic activity wasidentified within the human mediastinum region of the patient. Inaddition, this patient was to undergo routine cardiothoracic surgery. Atthe time of surgery the region was exposed, and within this region 5×5cm brown fat depot was dissected out and placed into sterile saline.

A brown fat depot was discovered in the mediastinum that contains apopulation of stem cells that express unique markers different thanthose found in other stem cells, including stem cells isolated fromwhite adipose depots. These newly identified stem cells were separatedfrom the mediastinum brown fat depot and cultured for greater than 20passages and still retained a normal karyotype. These cells also arecapable of undergoing adipogenesis (white and brown), osteogenesis, andchondrogenesis.

Example 6 Isolation of Cells from Human Brown Fat Depot Biopsy

The brown fat human depot isolated in Example 5 was washed 5-10 times insterile saline. The biopsy was cut using scissors until pieces areapproximately 0.1-0.5 cm in size. The material was washed 3 times bycentrifuging at 1200 RPM for 5 minutes. The material was then digestedusing either collagenase or dispase for no longer than an hour in ashaking water bath set at 37 C. After digestion was complete theenzymatic reaction was terminated by adding equal volume of completemedia. The material was then filtered using a 100 uM filter and thesingle cell suspension was washed 3 times with complete medium.

Example 7 Culture of Cells Isolated from Brown Fat Human Depot Biopsy

The single cell suspension described in Example 6 was plated onto cellculture flasks at a concentration of 5,000-10,000 cells/cm2. Afterapproximately 3-6 days the cells were ready for passaging (FIG. 2). Thecells after undergoing more than 10 passages showed a normal karyotype.RT-PCR analysis of the cultured brown fat cells demonstrated that theywere positive for CEBPB, UCP-1, UCP-2, PPARG, PGC-1, PRDM-16. It isimportant to note that cells isolated from white adipose depots werenegative for PRDM-16, and PGC-1. (FIG. 3). Accordingly, PRDM-16 and/orPGC-1 expression can be used to differentiate between white adiposecells and brown adipose cells. Moreover, CEBPB, UCP-1, UCP-2, PPARG,PGC-1, PRDM-16 can be used as markers to identify brown adipose cells.

Staining of the cells showed that they are positive for the stem cellmarkers HCAM and CD90 (FIGS. 4 A and B). The expanded cells alsoexpressed exosomes (FIG. 9). Flow cytometry of the cell populationdemonstrated that the cells were negative for the following cell surfacemarkers: CD34, CD19, CD86, HLA DR, Lin, CD106, CD80, CD 117. The cellpopulation was also low for SSEA4, and Stro-1. The cells were positivefor CD63, CD90, HLA ABC, CD105, CD73, CD166, CD 9, CD44 (FIGS. 5A-F)which are markers for undifferentiated brown adipose stem cells.

Example 8 Differentiation of Cells into Adipose (White and Brown)

Expanded cells were plated onto cell culture plates and supplementedwith adipogenic differentiation medium (Life Technologies). The mediawas changed every 3 days and differentiation was allowed to continue for14 days. After 7 days in induction media, fat droplets were visiblewithin the culture (FIG. 6), indicating that the cells had begundifferentiating into adipose cells.

Staining with oil red O demonstrated positive differentiation intoadipose cells (FIG. 7).

Identification of positive brown fat droplets in the differentiation wasobserved by the accumulation of smaller fat droplets (FIG. 7B) comparedto those found in cells differentiated from cells derived from white fatdepots, which produce larger fat droplets (FIG. 7A).

In another embodiment stem cells were plated in 6-well dishes at adensity of 50,000 cells/well. White or brown adipogenesisdifferentiation medium was added. For brown adipogenesis, FNDC5 wasadded 6 days post induction as previously described. 0.3% Oil Red O(Sigma Aldrich) was used for staining to detect intracellular lipidaccumulation.

Example 9 Chondrogenic and Osteogenic Induction of Cells

Expanded cells were plated onto cell culture plates and supplementedwith osteogenic and chondrogenic medium (Life Technologies). The mediawas changed every 3 days and differentiation was allowed to continue for21 days. Confirmation of differentiation into chondroblasts andosteoblasts was observed by staining of the cells with alizarin red orosteocalcin antibody and alcian blue (FIGS. 8 A and B). These dataindicate that progenitor cells isolated from brown fat depots maintainthe ability to differentiate in to a variety of cell types other thanwhite adipose or brown adipose.

Example 10 Therapeutic use of Brown Fat Progenitor Cells

NOD-SCID mice are severely immune deficient and are used as a modelsystem to test human cell-based therapies. Mice were raised on highfat/high carbohydrate chow throughout the test period. Mice were splitinto two groups: 10 untreated mice served as controls and 10 micereceived a single subcutaneous injection underneath the dorsal skin ofabout 1 million human brown fat progenitor cells cultured as describedin examples 5-9. The following analytes were measured for four monthsfollowing injection: plasma glucose, triglycerides, cholesterol, leptin,and adipon. Body weight also was monitored. Baseline is 23+/−5 g in thecontrol group and 25+/−7 g in the treated group. The control groupreceived a sham injection of carrier solution (DPBS) with no cells.

Compared to the control group that received a sham injection of carriersolution only (DPBS) with no cells, the experimental cohort whichreceived a dose of 1,000,000 cells demonstrated a decrease in plasmaglucose, triglycerides, cholesterol, and weight gain over a four monthmonitoring period. Adiponectin and leptin levels also correlated toimproved levels due to the having received the cell dose. This is insharp contrast compared to the untreated control mice that received thesham carrier solution (FIGS. 10-12).

EQUIVALENTS

It is to be understood that while the disclosure has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1-16. (canceled)
 17. A method of differentiating a stem cell into abrown fat cell, the method comprising: contacting a stem cell with amedium not comprising an animal product, thereby generating a brown fatstem cell.
 18. The method of claim 17, wherein the medium comprises anagent selected from the group consisting of: insulin, dexamethasone,isobutylmethylxanthine, indomethacin, T3, rosiglitazone, FNDC5 andcombinations thereof.
 19. The method of claim 17, wherein the mediumcomprises insulin, dexamethasone, isobutylmethylxanthine, andindomethacin.
 20. The method of claim 17, wherein the brown fat stemcell expresses PRDM-16, PGC-1, or a combination thereof.
 21. The methodof claim 17, wherein the brown fat tissue is from a sample obtained froma subject.